ASSAY DEVELOPMENT IN DRUG

DISCOVERY
Indira Padmalayam, Ph.D.

Drug Discovery Division, Southern Research Institute,

Birmingham, Alabama 35205
 The Drug Discovery Process: The Assay Development Stage
Chemistry
Structure-Activity Pre-clinical
“Hit” Relationship (SAR) GLP-Tox
validation bioavailability (PK, ADME), Clinical
toxicity
In vivo efficacy

Assay Lead Lead

Target Target
HTS Identification Validation Development
HTS Identification Optimization Development

Assay development
Primary assays
Secondary assays

Target Target Assay Lead

Non-HTS Identification Validation Development Optimization Development
Assay development: A critical part of the “hit” discovery process

Primary assays Secondary assays

“HITS”

A “hit” is a compound which

has the desired activity in a
compound screen and whose
activity is confirmed upon
retesting

Adapted frrom: Hughes, J.P. et.al. (2011), British Journal of

Pharmacology, 162 1239-1249
 Assay
Development

Pre-
Why is this a
bottleneck?
Assay Development=months
HTS=weeks

Roadblocks to faster assay development

Drug Discovery World, Summer 2010

 Topics to be covered

• Paradigms in Drug Discovery

• Significance of Assay Development in Drug Discovery
• Assay types
• Assay formats
• Optimizing assays for HTS
Factors to be considered (will be covered in detail in HTS lecture)
• Assay development examples
 Paradigms in Drug Discovery

Physiology-based Target-based
Target is unknown Known target

Physiological/phenotypic read-outs Read-outs are based on activity or expression of target

Cell-based assays Biochemical or Cell-based assays

The two paradigms are not mutually exclusive: Drug Discovery programs can use two-pronged
approach.
 Physiology-based drug discovery

Example : Viral CPE (Cytopathic Effect) assay

Influenza CPE assay - Preliminary CV Plate

140000

120000 Green: uninfected cells

Red: host cells+virus
100000 Blue: host cells+virus treated with
an antiviral compound, Ribavarin
Host cell viability

80000

60000

40000

20000
1
8
0
S1
S3
S5
S7

15
S9

S11

S13

S15

S17

S19

S21

S23

Slide:courtesy, Jim Noah, SRI

Target-based Drug Discovery

 Enzymes:
•Kinases
Receptor Tyrosine Kinase
Non-receptor tyrosine
kinase
Serine Threonine kinase
•Phosphatases
•Proteases:
Serine proteases
Zinc proteases
 Receptors:
Ion channel receptors
GPCRs
Nuclear receptors

Science, 2000
 Assay Development for Drug Discovery

 Target identified Assay Development HTS

and Validated

To ensure successful Assay Development:

-Ask the right “question” (the biological problem being addressed)
-Make sure that the “wording of the question” is correct (choose the correct assay, read-out and sequence of
assays).
Key Questions:
1. Are we trying to inhibit or activate the target?
2. What function of the target are we trying to regulate?
3. What are the types of assays that are available to measure the function?
4. Primary assay vs Secondary assays
5. Which assay will work best in terms of translatability to HTS
6. Which are the post-HTS assays that will drive Structure Activity Relationship (SAR)
 Key Considerations in Assay Development

The three “Rs”

-Relevance
-Robustness
-Reliability/Reproducibility

Practicality/Feasibility

Cost

Automation

“The quality of an assay determines the quality of the data: compromising on assay development
can have substantial downstream consequences”
 Types of Assays
Assays in Drug Discovery

Biochemical assays Cell-based assays

Target-based Phenotype-based
 Measure function of a purified target  Measure function of the target in the
Activity assays: Enzymes (e.g. kinases, proteases) context of the cell
Binding assays: Receptors -Transcriptional read-outs, second messenger levels,
(e.g. Nuclear receptors, Kinase receptors, ion cell viability (cell death/apoptosis), proliferation
channels, GPCRs)
 Measure expression of the target
 Identify compounds that modulate activity / mRNA levels, protein expression and localization
binding of the target protein
 Provide a functional read-out of
Recombinant (engineered) proteins, proteins isolated compound activity (as a functional consequence
from crude cell lysates of target engagement)
Monitor a surrogate read-out
 Examples: reporter assays, viability assays,
 Examples: Kinase/ATPase assays, GPCR and ion channel assays, qPCR
protease assays, protein interaction assays
 Biochemical versus Cell-based assays
Biochemical assays Cell-based assays
 Advantages:  Advantages:
-Simple -More physiological, amenable to systems approach
-More consistency -Can simultaneously assay for compound properties
-Direct measurement of target engagement (membrane permeability, toxicity, off-target effects)
-Can measure compound characteristics
such as Kd, Ki etc.
-Increased specificity of compounds
 Disadvantages:
-Complex
 Disadvantages: -High rate of noise
-May be non-physiological -Exclusion of less soluble/permeable compounds
-Not possible to determine compound
properties such as membrane permeability,
toxicity, off-target effects
 Causes of Assay Variation

Biochemical Assays Cell Based Assays

Same as for biochemical assays
Plus:
• pH
• Temperature
• Cell culture plastics
• Ion concentration
• Culture media
• Reagent Solubility
• Culture conditions
• Reagent Stability
• Serum
• Reagent Aggregation
• Cell cycle
• Order of reagent addition
• Passage number
• Instrumentation
 Common Assay formats

• Fluorescence
• Luminescence
 Fluorescence-based assays
 Based on excitation of a fluorophore 1 to 10 nano secs
 Variety of assays using fluorescence
- Simple assays where protein of interest is
conjugated to fluorophore, or where the
protein of interest generates a fluorescent
product
- Reporter assays

- Advantages: High sensitivity, ease of set-

up and operation
- Disadvantages: Prone to false positives
due to auto fluorescence of compound

Examples:
- FRET, TR-FRET
- Fluorescence polarization (FP)
 Fluorescence-based assays (contd.)
FRET: Florescence Resonance Energy Transfer

• Principle: Two fluorophores: Donor and Acceptor

-Based on transfer of energy between donor and acceptor
-Distance is critical
Far: No energy transfer no FRET
Close: Energy transfer from donor to acceptor FRET
• Use: Protein-protein, antigen-antibody, DNA-DNA, DNA-
protein
• Advantages:
-Homogenous assay format
-Reduced assay time and cost (HTS friendly)
• Disadvantage: Half life: 1 to 10 nano secs

-Short half life of fluorophore results in high background

 Fluorescence-based assays (contd.)
TR-FRET: Time Resolved-Florescence Resonance Energy Transfer

• “Improved”version of FRET
- Uses long lived fluorophores and time-
resolved detection to reduce backgound
-Rare earth elements (Lanthanides):
Samarium (Sm), Europium (Eu),Terbium
(Tb), and Dysprosium (Dy)

• Advantage:
-Low background; better signal to noise
• Disadvantage:
-Lanthanides have poor ability to absorb
light, so have to be complexed with
organic moieties that can harvest light
and transfer it to them.
 Fluorescence-based assays (contd.)
Fluorescence Polarization (FP) assay
 Principle:
Small, unbound fluorophore: fast rotation,
light is emitted in a plane different from
excitation light.
Fluorophore bound to protein: Slow rotation,
light is emitted in the same plane as
excitation light.
 Applications: Study molecular interactions
e.g. protein-protein, receptor-ligand, DNA-
protein, tyrosine Kinase assays.
 Advantages: Highly sensitive (low
picomolar range), homogenous assays,
multiple measurements can be made on the
same sample, because there is no change
in samples during the assay.
 Disadvantage: More optimization of assay
may be needed to ensure saturation of all
target binding sites with the fluorophore-
labeled ligand (to ensure displacement by
unlabeled ligand)
 Fluorescence-based assays (contd.)

FP assay
ATP binding pocket

Cy3 labeled FP
compound read

Un-labeled compounds

Screen
 Luminescence-based assays
Example: Luciferase reporter assay:
• Chemical reaction produces light
• Bioluminescence is the production and
emission of light by a living organism (e.g.
luciferase by firefly)

Luciferase
Luciferin + ATP Luciferyl adenylate+PPi

Luciferyl + O2 Oxyluciferin +AMP +Light

Adenylate
Examples of biochemical assays
 Assay for kinase activity

ADP Hunter assay

Fluorescent
product
Kinase target
Substrate Phosphorylated Substrate+ ADP

Inhibitors

Kinase target Fluorescent

Substrate Phosphorylated Substrate+ ADP product

Reduced signal
 Assay for protein-protein interaction
AlphaScreen technology
Amplified Luminescent Proximity Homogenous Assay

1O
2
Excitation at Emission at 520-
4 µsec 620 nm
680 nm Alpha
Alpha Donor
A B Acceptor
Bead
Bead
4 µsec

200 nm
 Alphascreen Assay: Measuring Tau-Fyn interactions in Alzheimer’s disease

• Interactions between Tau and Fyn are implicated in Alzheimer’s disease

• Donor bead labeled with glutathione
– Binds to Fyn-GST
• Acceptor bead chelated to nickel
– Binds to a His-tag on Tau protein

Figure: Courtesy Erik Roberson, ADDA Tau-Fyn project

Examples of cell-based assays
 Cell based assays
• Proliferation
Cell Proliferation
Fluorescent dyes (non-specific dye)

• Viability
-Assays that measure ATP Influenza CPE assay - Preliminary CV Plate

content (Cell Titer Glo), viral CPE 140000

(bioluminescence-based)
120000

100000
Cell viability
-Apoptosis assays 80000 (CPE assay)
(bioluminescence/fluorescence) 60000

40000

• Migration 20000
1

-Scratch assay
8
0
S1
S3
S5
S7 15

S9

S11

S13

S15

S17

S19

S21

S23
• Reporter Gene assays
Cell migration
-Transcriptional activity/expression
“scratch assay”
 Assays for G protein-coupled receptors (GPCRs)

• GPCRs are the targets for majority of

best-selling drugs and approx 40% of
prescription drugs (e.g. Zantac,
Clarinex, Zyprexa)
• GPCRs are proteins with 7
transmembrane domains
• Assay will depend on the type of GPCR
being targeted. 3 type of G proteins
(Gs, Gi/G0 and Gq)
• Non-G protein mediated responses
(β-Arrestin recruitment)
• Assays are designed to measure
second messengers such as cAMP,
Ca2+
• Measurement of proximal or distal
responses
• HTS friendly assays
 High Content Screening
High content screens:
• Facilitates validation of effects of
compounds at the cellular and subcellular
level (expression, localization,
morphological changes in cells)
• Macromolecules (e.g. proteins, RNA) are
labeled with fluorescent tags
• Technology uses automated digital
microscopy, flow cytometry and IT-
systems for analysis and storage of data
• Slower than HTS
• Popular as secondary screens in drug
discovery programs
 High Content Screening in Drug Discovery
Example 1: Cell migration assays: Oncology
Cytochalasin-D concentration response on cell migration

10µM 1µM 0.1µM 0.01µM

MDA-MB-231 breast cancer cells were co-cultured with 3T3L1 fibroblasts and treated with Cytochalasin-D, an inhibitor of actin
polymerization. Migration of cells through the matrigel layer was measured at various heights of the Z stack

Cytochalasin-D 48hrs 1
IC50 = 0.12uM
100
0.8
Cell Invasion Ratio

75
0.6
% Inhibition

50
0.4
25
0.2
0
0.001 0.01 0.1 1 10 100 0
Concentration (uM)
 High Content Screening in Drug Discovery
Example 2: Neurite Outgrowth Assay: Alzheimer’s disease
Aβ concentration response on neurite outgrowth

DMSO 5 nM 20 nM 80 nM 0.32 µM 1.25 µM 5 µM 10 µM 20 µM

Mean total neurite length per cell (µm) Mean length of longest neurite per cell (µm)
200

800
700 150
600
500
400 100
300
200 50
100
0
0
Assay development: From the bench to HTS

When you go
From This To This

THE RULES CHANGE

Everything is done in Microtiter Plates
96, 384, 1536, 3456

96-well 384-well 1536-well 3456-well

100-200µl 25-50µl 4-10 µl 1-2 µl
 Assay development: From the bench to HTS (HTS 101)
Assay development: From the bench to HTS

• What are you aiming for in an HTS assay:

• To have a reasonable chance, to believe the results of a single
determination, i.e. one well

• For that you need:

• Reproducibility from well to well
• Reproducibility from assay plate to assay plate
• Reproducibility from day to day

• How do you know if your assay is ready for HTS?

 ASSAY QC PARAMETERS

• “Z” factor: Key measure of readiness of an assay for HTS

3SD of sample + 3SD of control Z > 0.5
Z= 1 –
mean of sample – mean of control

• Coefficient of variance (CV) : Measure of dispersion % CV =SD X 100

Mean

• Signal to background (S/B) and Signal to noise (S/N): Measures of

signal strength
S/B = Mean of sample S/N = Mean of sample- Mean of control
Mean of control Standard Deviation of control

Will be covered in HTS lecture next week

ASSAY DEVELOPMENT CASE STUDIES
Case 1: Identification of a small molecule inhibitor of mitotic spindle bipolarity

Primary assay: Phenotype-based Counter screen Secondary assay: To select mitosis inhibitors

Identification of
Monastrol, inhibitor
of mitotic kinesin, Eg5

Mayer, TU eta al, Science 286, 971-974 (1999)

 CASE 2: Identifying compounds that inhibit TXNIP expression
ADDA project: TXNIP as a target for diabetes
TXNIP = Thioredoxin-interacting protein
Primary HTS assay: Luciferase reporter assay with TXNIP
promoter

Assay development HTS primary screen: 10K pilot screen followed by 300K
Primary and secondary assays
full screen
(single dose)
 3000 “Hits”
HTS primary screen
Hit validation and determination of potency
(single dose)
1. Cytotox assay to eliminate compounds with toxicity
2. Concentration response in primary assay for potency (IC50)
“Hits”
3. Chemistry analysis to identify scaffolds of interest

Hit validation and determination  1258 “Hits”

of potency (IC50)
(counter screens, concentration
response curves)
Counter screen: To eliminate non-specific luciferase inhibitors
651 “Hits”

Secondary assay: TXNIP qPCR

Secondary phenotypic assays 157 “Hits”

Chemistry analysis

37 “Hits”
13 “Hits”
Test in other confirmatory assays at UAB
 CASE 3: Identifying compounds that activate HO-1 expression
ADDA project: HO-1 as a target for multiple conditions
(chronic kidney disease, transplant rejection) Primary HTS assay: Luciferase reporter assay with HO-1
HO-1 = Heme Oxygenase-1 promoter containing enhancer element

Assay development HTS primary screen: 10K pilot screen followed by ~155K
Primary and secondary assays full screen
(single dose)
 2000 “Hits”

HTS primary screen Hit validation and determination of potency

1. Counter screen using a mutant form of the HO-1 promoter
(single dose)
2. Cytotox assay to eliminate compounds with toxicity
2. Concentration response in primary assay for potency (IC50)
“Hits”
3. Chemistry analysis to identify scaffolds of interest

Hit validation and determination  747 “Hits”

of potency (IC50)
(counter screens, concentration Chemical clustering analysis
response curves)
28 “Hits” representing six chemical clusters

Secondary assays: HO-1 qPCR and Western blot

Secondary phenotypic assays
“Analoging”: selection and purchase of analogs of hits

80 Compounds

Test in other confirmatory assays (qPCR and In Cell Western)

 NCGC Online Resources
 The National Chemical Genomics Center provides a
comprehensive online manual for assay development and
validation with HTS in mind.
For more information go to:
http://assay.nih.gov/assay/index.php/Table_of_Contents

 Manuscript helpful for HTS Assay Development:

Inglese, J. et.al. (2007), Nature Chemical Biology 3(8) 466-479
“The subject is not exhausted but we
are.”
George Bernard Shaw